Heraclitus.uth.gr

Materials and methods: Patient samples were collected with informedconsent, and disease activity quantiﬁed by the SELENA-SLEDAI index.
Multispectral imaging ﬂow cytometry was performed using an Amnis
Apoptotic parasites silence macrophages by misusing the
ImageStreamX instrument. LC3-positive autophagosomes were enu-
merated in viable, non-apoptotic cells using the Bright Detail Intensityalgorithm implemented in IDEAS 6. Autophagic flux was determined
P. Crauwels,* S. Gottwalt,* F. Ja¨ckel,* M. Thomas,* E. Bank,*
by incubation with chloroquine. As an alternative measure of
P. Walther, M. Bastianà & G. van Zandbergen*
autophagy, uptake of the novel autophagosomotropic dye CytoID
*Immunology, Paul-Ehlich-Institut, Langen, Germany, Electronmicros-
(Enzo) was analysed using conventional flow cytometry. Autophagy
copy Facility, University Ulm, Ulm, Germany, Veterinary Medicine,
was assayed in negatively selected B cells stimulated with combinations
of anti-IgM and anti-CD40 antibodies, and interferon-a.Results: Autophagy was signiﬁcantly increased in the CD19+ B cells of
Purpose/Objective: An appropriate T cell response to Leishmania
patients with SLE compared with healthy controls
(Lm) infection is critical for an effective immune response. Human
(P = <0.001, n = 22 patients, 15 controls), and there was a positive
macrophages (MF) can present antigen to T lymphocytes and at the
correlation with SLEDAI score (r = 0.67, P = <0.002). There was
same time serve as host cells. Upon macrophage infection the virulent
however, no association in CD4+ T cells (P = 0.49). There was no
inoculum of Lm promastigotes consists of apoptotic and viable
statistical evidence of confounding due to patient age or medication
promastigotes. The viable promastigotes enter a maturing phagolyso-
use. Assessment of autophagic ﬂux using the autophagosome-lysosome
some were they can survive and grow as amastigotes; the fate of
fusion inhibitor chloroquine revealed an accumulation of autophag-
Materials and methods: In this study, we hypothesize that the
Analysis of ex vivo viable, annexin V negative human B cells
apoptotic promastigotes use the MFs«autophagy machinery to down
demonstrated a signiﬁcant increase in autophagy in unstimulated
regulate MF antigen presentation and T cell activation.
compared with anti-IgM stimulated cells, with further decreases
Results: Upon promastigote uptake by human primary MFs, we
observed with the addition of anti-CD40 and interferon-a.
found apoptotic promastigotes to enter a compartment positive for the
Conclusions: The process of autophagy has not been previously
autophagy marker LC3. This LC3 compartment matured over time and
examined in ex vivo human B cells from patients with systemic
became LAMP positive. 24 h later the compartment resolved after
autoimmune disease. We demonstrate that autophagy is enhanced in
highly efﬁcient parasite degradation. When co-incubated with autol-
this context. Given our in vitro data, we may advance the hypothesis
ogous T lymphocytes, MFs infected with viable promastigotes induced
that autophagy is acting as a survival mechanism for auto reactive B
a strong CD4-positive T cell proliferation. Compared to viable
cells lacking adequate survival signals. An alternative explanation
parasites a signiﬁcantly lower T cell reactivity was observed in response
requiring further investigation is that autophagy is acting to promote
to MFs inoculated with apoptotic or a mixed population of apoptotic
presentation of self-antigens by B cells. Autophagy is readily inhibited
and viable parasites. Subsequently, preliminary results suggest that only
by many common pharmaceutical agents and may therefore represent
in the presence of apoptotic promastigotes and human T cells Lm
infection could be sustained in human MF over a period of 7 days.Conclusions: We found that apoptotic promastigotes enter a matur-ing LC3 compartment. Our data suggest that degradation of parasites
in this compartment could be involved in a down regulation of T cell
Role of autophagy in the immunopathogenesis of leprosy
activation. We now further investigate and characterize the prolifer-ating T cell subsets and how the autophagy machinery and apoptotic
B. J. Andrade Silva,* P. R. Andrade,* T. P. Amadeu,* V. Diniz,
promastigotes may dampen immune responses in human primary
S. Coˆrte-Real, V. C. Valentim,* H. Ferreira,* J. A. C. Nery,*
E. N. Sarno* & R. Olmo Pinheiro**Leprosy Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil,
Structural Biology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro,
Autophagy is activated in the B cells of patients with SLE andcorrelates with disease activity
Purpose/Objective: Leprosy is a chronic infectious disease that canpresent different clinical forms and there is evidence that the estab-
A. J. Clarke, U. Ellinghaus & T. J. Vyse
lishment of different clinical forms is driven by host innate mecha-
Medical and Molecular Genetics, King’s College London, London, UK
nisms. Macrophages from tuberculoid (BT) and lepromatous (LL)patients seem to have a different behavior in relation to the myco-
Purpose/Objective: Autophagy is increasingly appreciated as an
bacteria. While in LL patients there are highly infected macrophages, in
important immune surveillance and effector mechanism, but under-
BT rare or few bacilli are found. Electron microscopy studies showed
standing of its dynamic function in human autoimmune disease is
the presence of phagosomes with double membrane in macrophages
limited. We sought to evaluate its role in the B and T lymphocytes of
exposed to M. leprae (ML), suggesting a possible involvement of
patients with SLE compared with healthy controls.
Ó 2012 The Author(s) Ó 2012 Blackwell Publishing Ltd, Immunology, 137 (Suppl. 1), 185À772
506 Poster Session: Myeloid Cell Development
mechanism against liver injury. This study was scheduled to examine
Modiﬁcation of a single lysine in a CYP2E1 epitope induces
the role of SMAD7 in liver inﬂammation, ﬁbrosis and the possible
effect of antiviral treatment.Materials and methods: Liver biopsies from 67 patients with hepatic
D. Njoku,* J. Cho, L. Kim, L. Strouss, E. McCarthy, K. Gilbert,
diseases were studied: (1) 18 with chronic HCV hepatitis (CHC); (2)
19 with chronic HBV hepatitis at diagnosis (CHB/d); (3) four with
Johns Hopkins University, Anesthesiology Pathology Pediatrics, Balti-
CHB after antiviral treatment and relapse (CHB/non-r) (4) 14 with
more, MD, USA, Johns Hopkins University, Anesthesiology, Baltimore,
CHB after antiviral treatment response and remission for >5 years
MD, USA, àJohns Hopkins University, Biochemistry, Baltimore, MD,
(CHB/r); (5) 12 with non alcoholic fatty liver disease (NAFLD). Three
USA, §Johns Hopkins University, Pathology Molecular Microbiology and
liver samples with a mild increase of aminotransferases but without
histological changes, served as controls. Histological activity index and
Purpose/Objective: Key steps in the pathogenesis of immune-medi-
staging of ﬁbrosis were also assessed. RNA was extracted and cDNA
ated drug-induced liver injury (Im-DILI) have not been identiﬁed.
was synthesized using standard protocols. mRNA expression of TGFb
After receiving halogenated anesthetics, anti-seizure medications,
isoforms (TGFB1, 2, 3), activins (A, B, C, E), ALK4, ALK5, SMAD
antibiotics or non-steroidal anti-inﬂammatory drugs, susceptible pa-
molecules (SMAD2, 3, 4, 7), and CTGFwas examined using quanti-
tients develop Im-DILI thereby increasing their morbidity and often
tative real time PCR. Statistical analysis was performed using SPSS and
their mortality. In anesthetic Im-DILI patients, granulocytic hepatitis,
P values < 0.05 were considered signiﬁcant.
triﬂuoroacetyl chloride (TFA) and IL-4-mediated cytochrome P4502E1
Results: Patients with CHB/r exhibited a signiﬁcant increase of
(CYP2E1) IgG4 antibodies support the diagnosis, while CYP2E1 epi-
SMAD7 and ALK4 mRNA expression compared to CHB/d patients,
topes responsible for the pathogenesis of Im-DILI are unknown. We
and reduced levels of TGFB1, SMAD2, SMAD3, and CTGF. A
previously demonstrated a CYP2E1 epitope [Gly113-Leu133 (JHDN5)]
signiﬁcant increase of SMAD7 was also found in NAFLD patients
containing a single lysine that was recognized by sera from anesthetic
compared to untreated viral hepatitis patients and those who did not
DILI patients with specific MHC II haplotypes. We showed that
respond to any treatment. Moreover, NAFLD patients were presented
JHDN5 was recognized by splenocytes from mice with experimental
with elevated levels of TGFB1, TGFB3, INHbC, ALK5, and SMAD4.
Im-DILI induced by immunizations with liver proteins covalently al-
Considering the intensity of inﬂammation, SMAD7, ALK5, and INHbC
tered by TFA, a drug hapten formed during metabolism of halogenated
exhibited a signiﬁcant increased expression from absent to minimal
anesthetics. We hypothesize that covalent modification of a single
inﬂammation with a gradual reduction as inﬂammation exacerbates.
lysine in JHDN5 induces IL-4-mediated, Im-DILI in BALB/c mice.
Conclusions: Our data indicate that in cases with low grade ﬁbrosis,
Materials and methods: JDN5 was modiﬁed by TFA (TFA-JHDN5)
as NAFLD (characterized by a lower incidence of severe liver
using the methods of Goldberger and Anﬁnisen. We conﬁrmed 81.5%
complications and ﬁbrosis progression) and CHB/r, SMAD7 overex-
modiﬁcation of JHDN5 using the method of Habeeb. BALB/c mice
pression might be a mechanism limiting the ﬁbrogenic effect of TGFb
were immunized with 100 lg of an unrelated CYP2E1 epitope or
suggesting that its induction may provide a target for novel therapeutic
JHDN5 ± TFA emulsiﬁed in CFA or CFA alone on days 0 and 7 and
killed on day 21. IL-4 deﬁcient (KO) mice were similarly treated with
This research has been co-ﬁnanced by the ESF and Greek national
CFA ± TFA-JHDN5. Histology scores, antibodies and cytokine levels
funds through the Operational Program ‘‘Education and Lifelong
were analyzed using MannÀWhitney U-test. A P value <0.05 was
Learning’’ of the NSRF À Research Funding Program: Heracleitus II.
Investing in knowledge society through the European Social Fund.
Results: TFA-JHDN5 induced more granulocytic hepatitis (P < 0.01)as well as anti-TFA and anti-CYP2E1 antibodies (P < 0.05) than CFA-immunized BALB/c or KO mice. Granulocyte and macrophage
attractants KC, MIP-2, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-1b
Peritoneal macrophage inﬂammatory proﬁle in cirrhosis is
and VEGF as well as IL-7 and IL-9 were elevated in BALB/c but not KO
dependent on the etiology and is related to ERK phosphorylation
livers (P < 0.05). Unmodiﬁed epitopes did not induce Im-DILI.
Conclusions: We conﬁrm that covalent modiﬁcation of a single lysine
M. Martı´nez-Esparza,* A. Tapia-Abella´n,* A. J. Ruiz-Alcaraz,*
in a CYP2E1 epitope induces Im-DILI in BALB/c mice with features
T. Herna´ndez-Caselles,* C. Martı´nez-Pascual, M. Miras-Lo´pez,
reminiscent of anesthetic Im-DILI in patients. Future studies of this
J. Such,à R. France´sà & P. Garcı´a-Pen
epitope may uncover unidentiﬁed mechanisms of Im-DILI from other
*Biochemistry and Molecular Biology (B) and Immunology, University of
drugs and help to develop targeted agents to either treat or prevent this
Murcia, Murcia, Spain, Hospital Universitario Virgen de la Arrixaca,
Unidad de Trasplante Hepa´tico, Murcia, Spain, àHospital Universitario,Unidad Hepa´tica, Alicante, Spain
Purpose/Objective: The aim of this work is to identify functional
Overexpression of SMAD7 protects liver from TGFb/Smad-medi-
differences in the inﬂammatory proﬁle of monocyte-derived macro-
phages (M-DM) from ascites in cirrhotic patients of different etiolo-gies, alcohol- and hepatitis C virus (HCV)-related cirrhosis, trying to
N. Argentou,* G. Germanidis, E. Apostolou,à T. Vasiliadis,§
extrapolate studies from liver biopsies to immune cells in ascites.
P. Sideras,à A. E. Germenis* & M. Speletas*
Materials and methods: We studied 45 patients with cirrhosis and
*Medical School, University of Thessaly, Larissa, Greece, AHEPA
non-infected ascites, distributed according to disease etiology, HCV
Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece,
(n = 15) or alcohol (n = 30). Cytokines and cellular content in ascites
Biomedical Research Foundation, Academy of Athens, Athens, Greece,
were assessed by ELISA and ﬂow cytometry, respectively. Cytokines
Hippokration Hospital, Aristotle University of Thessaloniki, Thessalo-
and ERK phosphorylation level from peritoneal monocyte-derived
macrophages isolated and stimulated in vitro were also determined.
Purpose/Objective: SMAD7 is a negative regulator of TGFb/activin
Results: A different pattern of leukocyte migration to peritoneal cavity
pathway. Recently, animal studies have shown that SMAD7 induction
and primed status of macrophages in cirrhosis is observed depending
ameliorates TGFb/Smad-mediated ﬁbrogenesis, suggesting a protective
on the viral or alcoholic etiology. Whereas no differences in peripheral
Ó 2012 The Author(s) Ó 2012 Blackwell Publishing Ltd, Immunology, 137 (Suppl. 1), 185À772

Posters_Abstracts of the European Congress of Immunology,p12, 5-8 September 2012, Glasgow, Scotland

Questions 4 This diagram shows some of the elements in Core curriculum 1 This extract from the Periodic Table shows the Look at the row from lithium (Li) to neon (Ne). a What are the elements in this group called? a What is this row of the Periodic Table called? b Chlorine reacts explosively with hydrogen. The b Which element in it is the least reactive? Why? Look

February 2004 By Merrill Goozner and Jeff DelViscio The Use of SSRIs in Children: An Industry-Biased Record American psychiatrists and other physicians have steadily increased their use of serotonin reuptake inhibitors (SSRIs) to treat children suffering from depression and other psychiatric disorders. One study found that between 1997 and 2000, pediatric use of SSRIs surged 18.